Background: We aimed to evaluate the feasibility of a non-contrast time-of-flight magnetic resonance angiography (TOF-MRA) protocol for the pre-procedural access route assessment of transcatheter aortic valve implantation (TAVI) in comparison with contrast-enhanced cardiac dual-source computed tomography angiography (CTA). Methods and Results: In total, 51 consecutive patients (mean age: 82.69 ± 5.69 years) who had undergone a pre-TAVI cardiac CTA received TOF-MRA for a pre-procedural access route assessment. The MRA image quality was rated as very good , with only four examinations rated as non-diagnostic. The TOF-MRA systematically underestimated the minimal effective vessel diameter in comparison with CTA /external iliac artery (EIA)/common femoral artery (CFA) MRA vs. CTA was 8.04 ± 1.46 vs. 8.37 ± 1.54 (p < 0.0001) and the left CIA/EIA/CFA MRA vs. CTA was 8.07 ± 1.32 vs. 8.28 ± 1.34 (p < 0.0001)). The absolute difference between the MRA and CTA was small . The overall correlation between the MRA and CTA measurements was very good (with a Pearson correlation coefficient of 0.87 (p < 0.0001) for the right CIA/EIA/CFA and a Pearson correlation coefficient of 0.9 (p < 0.0001) for the left CIA/EIA/CFA). The feasibility agreement between the MRA and CTA for transfemoral access was good ). Conclusions: The TOF-MRA protocol was feasible for the assessment of the access route in an all-comer pre-TAVI population. This protocol might be a reliable technique for patients at an increased risk of contrast-induced nephropathy. At present, transcatheter aortic valve implantation (TAVI) is the standard of care for patients with severe aortic stenosis and increased perioperative risks ,2,3. ComIn a previous study, we described the feasibility and accuracy of respiratory and ECG-triggered 3D non-contrast MRA for aortic annulus assessments in comparison with contrast-enhanced CTA . To dateThus, the aim of this study was to evaluate the feasibility and accuracy of a non-contrast time-of-flight magnetic resonance angiography (TOF-MRA) protocol for pre-procedural femoral access route assessments of transcatheter aortic valve implantation (TAVR) in comparison with contrast-enhanced cardiac dual-source computed tomography angiography (CTA).The potential candidates for inclusion in this study were all patients with severe symptomatic aortic stenosis who had been referred for CTA prior to a TAVI evaluation between February 2014 and March 2017. Patients were prospectively enrolled if none of the exclusion criteria were present. The exclusion criteria were a rejection of the MRA examination, the inability to remain in a supine position due to severe orthopnea or a reduced general state of health, the presence of metallic foreign bodies, a permanent pacemaker or severe claustrophobia. This prospective study was approved by the institutional review board and complied with the Declaration of Helsinki.CTA examinations were performed using a second-generation dual-source CT scanner .®, Bracco, Konstanz, Germany) was used as an iodinated contrast agent with a dual-phasic injection protocol , with bolus-triggering in the ascending aorta. For this purpose, a region of interest (ROI) was placed in the left atrium. Data acquisition began 7 seconds after the attenuation of the ROI reached 70 Hounsfield units (HUs) (a bolus tracking technique), with the retrospective ECG-gated data acquisition of the aortic root followed by a non-gated aortofemoral high-pitch spiral dual-source acquisition.In total, 50 mL of a commercially available contrast medium .All patients were examined using a 3T MRI system . Time-of-flight (TOF) MRA was used for the assessment of the femoral access route. The following scan parameters were applied: repetition time (TR) of 458 ms, echo time (TE) of 3.7 ms and flip angle of 50°. The slice thickness was 3.5 mm.Two experienced readers  independently evaluated the MRA measurements. They were blinded to the clinical findings, the medical histories of the patients and the CTA images. To avoid recall bias, the measurements for CTA were performed after a delay of two months; this was undertaken in consensus with the readers (T.K. and P.R.), who were blinded to the MRA measurements.Our study protocol for MRA focused on the iliofemoral vessels; thus, it did not include an assessment of the aorta. All measurements were obtained from the common iliac artery (CIA), the external iliac artery (EIA) and the common femoral artery (CFA) of the iliofemoral vessels on each side. The minimal effective diameter and area were assessed for each segment. Multiplanar reformation (MPR) was used for the measurements of the minimal effective diameter. Two planes were used to create a cross-sectional image that was orthogonal to the blood flow direction. The area was measured on the cross-sectional image at the site of maximal lumen narrowing . The minssection .The subjective image quality of TOF-MRA was graded by both observers using a semi-quantitative five-point scale. A grade of 5 indicated an excellent image quality with a high signal without artifacts; 4 indicated a good-quality image with a high signal without artifacts or only minor artifacts; 3 indicated a moderate-quality image with a weak signal, but the exclusion of relevant stenosis was possible; 2 indicated a low-quality image with a low signal or the presence of artifacts and where stenosis could not be ruled out; and 1 indicated a non-diagnostic image.t-test or a Mann–Whitney U test, as appropriate. The MRA and CTA measurements were correlated using Spearman’s or Pearson’s rank tests, as appropriate. Interobserver variabilities of measurements were calculated using the intraclass correlation coefficient (ICC) with a two-way model. A comparison of the feasibility of transfemoral access using MRA and CTA was performed using an agreement and Cohen’s kappa. The irr package (version 0.84.1) was used to calculate the intraclass correlation coefficient and Cohen’s kappa. A p-value below 0.05 was considered to be statistically significant.All statistical analyses were performed using R (version 4.0.4). The continuous variables were presented as the mean and standard deviation. Differences in measurements were evaluated using Bland–Altman plots (mean difference ± 1.96 SD) and tested using Student’s 2). The mean eGFR was 54.2 ± 20.8 mL/min/1.73 m2. The baseline characteristics are presented in The MRA was performed after the CTA for TAVI evaluations of 51 patients with severe aortic stenosis (31 women (60.8%), mean age: 82.69 ± 5.69 years). The mean time interval between the CTA and MRA was 1.43 ± 2.39 days. In total, 60.7% of patients demonstrated impaired renal function  of the hip. Similarly, when evaluating the left transfemoral access route, there were five patients with inadequate image quality; three were due to artifacts as a result of a hip TEP. For the evaluation of transfemoral access through the right CIA/EIA/CFA, the image quality (≥3) was adequate in 47/51 patients (92%); for the left CIA/EIA/CFA, the image quality was adequate in 46/51 (90%) patients. In 44/51 (86.3%) of patients, there was adequate image quality for a simultaneous evaluation of both the right and left sides .p < 0.0001) and the left transfemoral access route (CIA/EIA/CFA) MRA vs. CTA was 8.07 ± 1.32 vs. 8.28 ± 1.34 (p < 0.0001)) (p < 0.0001); left transfemoral access route (CIA/EIA/CFA) Pearson correlation coefficient: 0.9 (p < 0.0001))  MRA vs. CTA was 8.04 ± 1.46 vs. 8.37 ± 1.54 (0.0001)) . The abs0.0001)) . The sam0.0001)) . The int0.0001)) .p = 0.001)). There was one case of a short dissection of the left common iliac artery , the potential risks and benefits, as well as alternative imaging strategies, should be carefully considered before performing contrast-enhanced computed tomography. The guidelines recommend volume expansion using normal saline as a prophylaxis in these patients to prevent an acute kidney injury prior to the administration of intravenous iodinated contrast media [The risk of the development of an acute kidney injury after CTA prior to TAVI is higher for patients with pre-existing kidney disease and when higher volumes of an intravenous iodinated contrast agent are applied . Recentlst media . This rest media . These fAlthough the patient cohort in this study was one of the largest on the subject of using MRA for the assessment of iliofemoral access routes prior to TAVI, the number of study participants was low in comparison with the data available from studies using CTA. The study protocol for MRA did not include an assessment of the aorta; contraindications on the level of the aorta  could not be evaluated using MRA.Longer scan times when using MRA, especially TOF-MRA, might hinder its application for certain patients, especially older and more frail patients, where lying calmly in a supine position without moving for a long time might be difficult. Those patients might benefit from faster MRA sequences with shorter scan times, as provided by QISS-MRA.The non-contrast MRA evaluation of transfemoral access is a promising approach for patients with chronic kidney disease who are referred for TAVI. Whether this translates into better outcomes for these patients is unclear, as there are no randomized data currently available on this subject. MRA is a more time-consuming and expensive technique compared with CTA; a substantial benefit to the patient would need to be demonstrated to justify its use. Further prospective, preferably randomized, studies are required before non-contrast MRA can be recommended for broader applications. In this context, data from an ongoing randomized trial are eagerly awaited .The evaluation of iliofemoral access routes for TAVI using non-contrast MRA is feasible and reliable. Patients at risk of contrast-induced nephropathy might particularly benefit from such an approach. Whether this approach translates into better overall clinical outcomes after TAVI requires evaluation in future studies.


Sp-IR) from the mud crab Scylla paramamosain. Sequence analysis revealed that this receptor consists of a Fu domain, two L domains, three FN-III domains, a transmembrane domain, and a tyrosine kinase domain, classifying it as a member of the tyrosine kinase insulin-like receptors family. Our results also suggested that Sp-IR was highly expressed in the testis and AG in males. Its expression in the testis peaked in stage I but significantly decreased in stages II and III (p < 0.01). Next, both short- and long-term RNA interference (RNAi) experiments were performed on males in stage I to explore Sp-IR function in mud crabs. The results showed that Sp-vasa and Sp-Dsx expression levels in the testis were significantly down-regulated after the specific knockdown of Sp-IR by RNAi. Additionally, the long-term knockdown of Sp-IR led to a considerable decrease in the volume of seminiferous tubules, accompanied by large vacuoles and a reduced production of secondary spermatocytes and spermatids. In conclusion, our results indicated that Sp-IR is involved in testicular development and plays a crucial role in transitioning from primary to secondary spermatocytes. This study provided a molecular basis for the subsequent analysis of the mechanism on male sexual differentiation in Brachyuran crabs.Insulin-like androgenic gland hormone (IAG) is a key regulator of male sexual differentiation in crustaceans that plays important roles in secondary sexual characteristics and testicular development. As a hormone, IAG interacts with its membrane receptor to initiate downstream signal pathways to exert its biological functions. In this study, we isolated a full-length cDNA of an insulin-like receptor ( Sexual determination and differentiation are prominent topics in biological research. Shrimps and crabs, as representative groups of crustacean species, are valuable protein sources for humans and hold significant economic importance in the marine organism industry. It has been observed that there are distinct disparities in growth rate, individual size, and nutritional value between male and female individuals in certain species ,2. ThereCherax quadricarinatus), silencing the IAG gene resulted in the feminization of some male individuals, evidenced by a reduction in the number of spermatozoa, the degeneration of a significant portion of the testis, and the expression of the vitellogenin gene, which is typically associated with female reproduction [Lysmata vittata, which exhibits protandric simultaneous hermaphroditism, demonstrated that inhibiting IAG had significant effects. The knockdown of IAG resulted in the inhibition of both oogenesis and spermatogenesis, as well as the development of abnormal appendices masculinae [Macrobrachium rosenbergii, the removal of the AG from juvenile males led to sex reversal [Mr-IAG gene resulted in AG hypertrophy and the arrest of testicular spermatogenesis [M. rosenbergii [The androgenic gland (AG) is a unique endocrine gland found in male crustaceans, playing a crucial role in regulating gender differentiation, spermatogenesis, and the maintenance of male secondary sexual characteristics ,4. Previoduction . A recensculinae . Similarreversal . Additioogenesis . Furtherenbergii . By manienbergii ,14.Mr-IR) was isolated from the giant freshwater prawn M. rosenbergii [Mr-IR is widely expressed in various tissues of both male and female individuals. Knockdown of Mr-IR in male individuals through RNA interference (RNAi) did not affect their growth but impeded the development of spermatocytes and led to AG hypertrophy [Mr-IR in males using small interfering RNA (siRNA) induced sex reversal, indicating the involvement of Mr-IR in the regulation of sexual differentiation [Sagmariasus verreauxi, the tyrosine kinase insulin receptor (Sv-TKIR) is expressed in the male testis and antennal glands in both sexes [Fenneropenaeus chinensis, Fc-IAGR is specifically expressed in males. Prolonged suppression of Fc-IAGR gene expression leads to a significant decrease in the amount of sperm cells and results in the arrest of germ cells at the secondary spermatocyte stage [The insulin-like receptor (IR) is a transmembrane receptor belonging to the tyrosine kinase receptor superfamily. It is initially synthesized as a single chain and undergoes glycosylation and folding to form a mature protein, an α2β2 dimer, which plays a crucial role in transmitting the cascade signals of insulin-like peptides from the extracellular to the intracellular region ,16. The enbergii . Mr-IR iertrophy . Furtherntiation . In the th sexes . Notablyte stage .S. paramamosain in China, play a significant role in coastal fisheries in tropical and subtropical Asia [Mud crabs, particularly the dominant species cal Asia . In rececal Asia . In decacal Asia . HoweverS. paramamosain. Based on its expression profile in the testis, we proposed that Sp-IR may participate in testicular development and spermatogenesis as a putative IAG receptor. Subsequently, both short- and long-term RNAi experiments were performed to validate the function of Sp-IR in male S. paramamosain.In this study, we first cloned and identified an insulin-like receptor (IR) gene from the mud crab Sp-IR was successfully obtained through a combination of 3′ and 5′ RACE techniques and touchdown PCR. The mRNA sequence of Sp-IR in the testis of S. paramamosain was found to be 6538 bp in length (GenBank OQ361826). It consisted of a 306 bp 5′ untranslated region (UTR), a 559 bp 3′ UTR, and a substantial open reading frame (ORF) spanning 5673 bp. The ORF encoded a protein consisting of 1890 amino acids .To investigate the potential involvement of  stage I . Howevervasa, Dsx, Sxl, Tra, Dmrt, and Sox-5 genes [S. paramamosain, Sp-vasa was specifically expressed in the testis, and its mRNA was detected exclusively in germ cells [Sp-vasa gradually increased from spermatogonia to spermatocytes but was absent in spermatozoa, indicating its involvement in spermatogenesis before the formation of spermatozoa. Similarly, the expression pattern of the Dsx gene in the testis of F. chinensis is comparable to that of Sp-vasa [Fc-Dsx was shown to inhibit Fc-IAG gene expression, suggesting the involvement of Dsx in IAG-regulated spermatogenesis [Sp-vasa and Sp-Dsx as indicators of spermatogenesis.In studying sex determination and differentiation mechanisms in crustaceans, researchers often rely on homologous marker genes identified in model organisms, such as -5 genes . Vasa is-5 genes . It has -5 genes ,26,27. Irm cells . The exp Sp-vasa . Silenciogenesis . TherefoSp-IR knockdown in the testis, male crabs at stage I were subjected to RNAi by injecting Sp-IR dsRNA. The results demonstrated a significant reduction in the expression level of Sp-IR after Sp-IR dsRNA injection (p < 0.01). The knockdown efficiency reached up to 85.97% . In contrast, the injection of GFP dsRNA had no significant effect on the expression levels of Sp-IR, Sp-vasa or Sp-Dsx.To assess the effectiveness of o 85.97% A. Furthe Sp-vasa B and Sp- . The gene knockdown efficiency was calculated to be 75.83% (Sp-vasa (Sp-Dsx (Sp-IR dsRNA (p < 0.01). In contrast, the injection of GFP dsRNA had no notable effect on the expression levels of Sp-IR, Sp-vasa, or Sp-Dsx transcripts in the testis.To further investigate the role of e 75.83% A. Simila from the mud crab S. paramamosain, and through RNAi technology, we investigated the specific functions of Sp-IR in spermatogenesis and testicular development.The understanding of male sexual differentiation in crustaceans has been predominantly attributed to the role of IAG derived from the AG ,31. HoweSp-IR gene identified in the mud crab S. paramamosain was found to be 1890 aa in length. It consists of various structural domains, including a FU domain, two L domains, three FN-III domains, a transmembrane domain, and a tyrosine kinase domain, consistent with other members of the tyrosine kinase receptor superfamily [Sp-IR with IR genes reported in other species. Specifically, the sequence similarity of Sp-IR with the insulin-like receptor genes of S. verreauxi and F. chinensis in the tyrosine kinase domain exceeded 70% [Sp-IR and other decapod crustaceans. These findings suggest that IRs in crustacean species are highly conserved, and the identified Sp-IR gene may serve as a putative IAG receptor in the mud crab. However, further functional assays are necessary to confirm whether this putative receptor can indeed be activated by IAG.The erfamily . Multipleded 70% ,20. FurtSp-IR exhibited high expression levels in the testis and AG of male S. paramamosain, consistent with the findings reported in F. chinensis and S. verreauxi, where Fc-IAGR and Sv-IR were also highly expressed in the testis [M. rosenbergii, the IR gene showed high expression in the female ovary and antennal gland, while its expression was low in the male AG, cerebral ganglion, and thoracic ganglion of both male and female individuals [Sp-IR in the testis of S. paramamosain indicated its potential involvement in testicular development. The expression profile of Sp-IR mRNA during testicular development was investigated by qRT-PCR. The findings revealed that the expression of Sp-IR transcript was highly abundant at stage I but significantly decreased at stages II and III , suggesting that the regulation of spermatogenesis by insulin-like signaling in S. paramamosain is likely to be more complex.To further confirm the roles of  gametes . Indeed, gametes . Studies gametes . Knockdo gametes . Similarhinensis ,20. In Fte stage . In contSp-IR) in the mud crab S. paramamosain. Sp-IR was found to be highly expressed in the testis and AG and was involved in the transition of primary spermatocytes to secondary spermatocytes and testicular development. As a putative receptor for IAG, the findings provide valuable insights into the mechanism underlying male sexual differentiation in Brachyuran crabs. Further research is needed to elucidate the precise role of Sp-IR and its interaction with IAG in regulating sexual differentiation.In conclusion, this study identified and characterized an insulin-like receptor (Scylla paramamosain) were purchased from a local fisheries wholesale market in the Si-Ming District of Xiamen . The male individuals selected for the experiments exhibited healthy appendages and good vitality and were at the intermolt stage. Prior to the start of the experiments, the crabs were acclimated in tanks containing filtered seawater at a temperature of 27 ± 2 °C and salinity of 27 ± 0.5 ppt for 5 days and fed with fresh Manila clams (Ruditapes philippinarum) daily. Any uneaten bait was promptly removed, and the tanks were replenished with fresh seawater containing sufficient oxygen the following morning.Mud crabs , stage II , and stage III . The crabs were anesthetized by placing them on ice, and testis samples were collected from males at each stage (n = 4–5) for Sp-IR expression profile analysis using real-time quantitative PCR (qRT-PCR). The samples were then either immediately used for RNA extraction or stored in liquid nitrogen.Since the differentiation of spermatogonia and spermatocytes occurs before stage IV, the crabs in stages I to III were selected to analyze w/v) agarose gel electrophoresis. First-strand cDNA synthesis was conducted for subsequent experiments using RevertAid™ First Strand cDNA Synthesis Kit (Thermo Scientific).Total RNA was extracted from the samples using TRIzol  following the manufacturer’s instructions. The concentration and quality of the extracted RNA were determined using a Q6000 spectrophotometer . The integrity of the RNA was evaluated by performing 1.5%  following the manufacturer’s protocol. The PCR products obtained were separated by 1.5% (w/v) agarose gels, purified using an agarose gel purification and extraction kit , and ligated into a pMD19-T vector  for further sequencing. Then, the full-length cDNA sequence of Sp-IR was assembled from the obtained sequencing results. The primers utilized in this study are listed in A partial cDNA sequence encoding an insulin-like receptor was initially identified from the transcriptome database of Sp-IR were predicted using the ORF finder tool. The conserved domains of Sp-IR were predicted using the SMART website , while the transmembrane domain of Sp-IR was predicted using an online website . To determine the presence of a protein signal peptide, the amino acid sequence was submitted to the SignalP 5.0 Server . To assess the homology of the amino acid sequence of Sp-IR with those of other species, BlastX homology searches were performed using the NCBI database . Multiple sequence alignment of the tyrosine kinase domain was conducted using Cluster X2.0 and GeneDoc2.7.000 software. Phylogenetic trees were constructed using the neighbor-joining (NJ) method implemented in MEGA 7.0.26 software, with bootstrap sampling repeated 1000 times to assess the reliability of the tree.The open reading frame (ORF) and amino acid sequence of Sp-IR in male mud crabs at stage II, reverse transcription PCR (RT-PCR) was performed. The cDNA templates were generated from 11 different tissues, including the eyestalk ganglion, cerebral ganglion, thoracic ganglion, heart, muscles, hepatopancreas, stomach, epidermis, testis, androgenic gland, and Y-organ. PCR was performed using the following conditions: an initial denaturation step at 95 °C for 3 min, followed by 36 cycles of denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s, and extension at 72 °C for 30 s. The final extension step was performed at 72 °C for 10 min. Negative controls, using water as a template, were included to ensure the absence of contamination. The amplification of β-actin was used as the internal control for normalization purposes. After amplification, the PCR products were visualized by electrophoresis on a 1.5% agarose gel and imaged using a UV gel imager . The experiment was repeated thrice.To determine the tissue distribution of IR dsRNA and GFP dsRNA, was analyzed by electrophoresis on a 1.5% agarose gel to confirm the presence and integrity of the dsRNA. The concentrations of the dsRNA samples were then measured using a NanoDrop 2000 spectrophotometer from Thermo Fisher Scientific and stored at −80 °C until further use.To synthesize dsRNA, T7 and SP6 RNA polymerase from Takara were used under the following conditions: incubation at 37 °C for 2 h, followed by a heat inactivation step at 70 °C for 10 min, and a final incubation at 37 °C for 20 min. The synthesized dsRNA, including n = 10). The first group of crabs was injected with IR dsRNA (1 μg/g body mass) prepared in 100 μL crab saline. The second group received only 100 μL of crab saline, while the third group was injected with GFP dsRNA (1 μg/g body mass) prepared in 100 μL crab saline. Injections were performed once daily using a microsyringe  with a thin needle inserted into the arthrodial membrane at the fifth swimming leg. Approximately 48 h after the second injection, the crabs were anesthetized by placing them on ice. Testis samples were dissected and collected for gene expression analysis. Specifically, the expression levels of Sp-IR, Sp-vasa, and Sp-Dsx genes were assessed using quantitative real-time PCR (qRT-PCR).For this experiment, male mud crabs at stage I with a body mass of 36.72 ± 2.84 were used. Prior to injection, the crabs were temporarily housed in the laboratory for approximately 5 days. They were then randomly divided into three groups . The remaining crabs were divided into three groups (n = 30). The control group received an injection of 100 μL of crab saline. The other two groups were injected with Sp-IR dsRNA and GFP dsRNA (1 μg/g body mass) prepared in 100 μL of crab saline, respectively. The injections were performed once every five days, and on day 22 of the experiment, approximately 48 h after the fourth injection, the crabs were anesthetized by placing them on ice and then euthanized. Testis samples were dissected from the crabs for gene expression analysis of Sp-IR, Sp-vasa, and Sp-Dsx, using techniques such as qRT-PCR. Additionally, histological analysis of the testis was conducted through hematoxylin and eosin staining.To investigate the impact of β-actin gene was simultaneously amplified as an internal control to normalize the data. The primers used for the qRT-PCR are listed in −ΔΔCt method.The relative expression levels of the target genes were determined using qRT-PCR. The reactions were carried out on a 7500 Fast Real-Time PCR Detection System  with a total reaction volume of 20 μL. Each reaction included 2 μL of diluted cDNA, 10 μL of 2× PCR Master Mix with SYBR GREEN , 0.5 μL each of forward and reverse primers (1 mM), and 7 μL of water. The thermal profile for the qRT-PCR reaction consisted of an initial incubation at 95 °C for 3 min, followed by 40 cycles of amplification with the following conditions: denaturation at 95 °C for 10 s, annealing at 58 °C for 30 s, and extension at 72 °C for 30 s. Each sample was analyzed in triplicate, and the p < 0.05.The data are presented as mean ± SEM (standard error of the mean). Statistical analysis was performed using the SPSS v20.0 software. Levene’s test was performed to examine the homogeneity of data variance. If the variance was found to be homogeneous, significance analysis was performed using one-way analysis of variance (ANOVA), followed by Duncan’s test or Dunnett multiple comparison test. Statistical significance was set at


Carbon nanotube-based derivatives have attracted considerable research interest due to their unique structure and fascinating physicochemical properties. However, the controlled growth mechanism of these derivatives remains unclear, and the synthesis efficiency is low. Herein, we proposed a defect-induced strategy for the efficient heteroepitaxial growth of single-wall carbon nanotubes (SWCNTs)@hexagonal boron nitride (h-BN) films. Air plasma treatment was first performed to generate defects on the wall of SWCNTs. Then, atmospheric pressure chemical vapor deposition was conducted to grow h-BN on the surface of SWCNTs. Controlled experiments combined with first-principles calculations revealed that the induced defects on the wall of SWCNTs function as nucleation sites for the efficient heteroepitaxial growth of h-BN. Both controllability and efficiency were improved significantly. Our work provides an efficient approach to the preparation of SWCNT-based derivative macrostructures, which may facilitate their applications in a wide range of areas.In this study, we developed a defect-induced heteroepitaxial method to efficiently synthesize SWCNT@h-BN films. Freestanding high-quality SWCNT films prepared by the floating catalysts CVD (FCCVD) method were used as a growth template. Air plasma treatment was performed to introduce defects on the tube walls that serve as nucleation sites in the following heteroepitaxial growth of h-BN by atmospheric pressure CVD (APCVD). Macroscopic SWCNT@h-BN films were obtained, and their growth mechanism was elucidated by combining experimental investigation and first-principles calculations. The microstructure of the SWCNT@h-BN films was tuned by adjusting the density of defects on the SWCNT template. Defects were controllably introduced by air plasma, and more sites were supplied for the nucleation of h-BN. Furthermore, the spSWCNT films were synthesized by an injection FCCVD method . Freesta4H14BN, 97% Sigma-Aldrich, Burlington, MA, USA) as a precursor. A schematic of the APCVD setup is shown in 4H14BN was loaded upstream and heated to 90 °C by a heating belt, and 300 sccm of Ar was introduced as a carrier gas. The reaction temperature was set to be 1000 °C, and the growth time was adjusted in the range of 5–180 min. After growth, the CVD furnace was cooled down to ambient temperature naturally under the protection of an Ar flow.Before the CVD growth of h-BN, air plasma treatment was performed to controllably introduce defects on the wall of SWCNTs . Briefly2 F20, Hillsboro, Oregon, USA, operated at 200 kV) was used to characterize the microstructure of the SWCNTs and SWCNT@h-BN films. High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) image and electron energy loss spectroscopy (EELS) mapping of SWCNT@h-BN were acquired using a spherical aberration-corrected TEM .Transmission electron microscopy  spectra of all samples were collected from spectrometer  in the range of 400–4000 cm−1. X-ray photoelectron spectroscopy  was used to analyze the chemical composition of surface layers using a pass energy of 50 eV with an energy step size of 0.1 eV. The collected spectra were calibrated by the standard C 1s peak located at 284.6 eV.The Raman spectra of SWCNTs and SWCNT@h-BN films were collected by micro-Raman spectroscopy . The ratios of IFirst-principles calculations based on the density functional theory implemented in the Vienna ab initio simulation package (VASP) ,32 with 2 bond of h-BN [2 hybridization of SWCNT and h-BN increased the interaction and triggered efficient epitaxial growth. Additionally, the formation of sp2 hybridization would improve the capacity of phonon transformation, realizing excellent thermal conductivity.To characterize the chemical composition of the layer structure coated on the SWCNT surface, EELS was acquired in the STEM mode with an energy resolution of 2 eV. The low-magnification annular dark-field (ADF) image a showed  of h-BN ,37. The G/ID) is widely used to evaluate the crystallinity of SWCNTs. The IG/ID of the original SWCNT film was 133, indicating its high quality. After air plasma treatment, the IG/ID value decreased to 4.6, demonstrating that abundant defects were introduced. Since the location of the D band (around 1350 cm−1) for sp3 carbon and E2g (around 1366 cm−1) peak for h-BN is very close [−1  Raman peaks collected from pristine SWCNTs, plasma-treated SWCNTs, and SWCNT@h-BN are shown in ry close , to distlose [−1 , and theN growth h. The peB-N bond ,17.To characterize the elemental stoichiometry and bond characteristics of the SWCNT@h-BN, XPS measurement was also performed. As shown in 4H14BN tended to adsorb on the defect sites and to form covalent bonds with C (We proposed a defect-induced heteroepitaxial growth mechanism of SWCNT@h-BN, as shown in s with C b,c. As ts with C  by usings with C c,d.To investigate the influence of growth parameters on the morphology of SWCNT@h-BN, we performed APCVD under different conditions. APCVD was first performed at 900 °C, 1000 °C, and 1100 °C for the growth of h-BN. As shown in To verify the proposed defect-induced growth mechanism, we performed a control experiment by using SWCNTs without plasma treatment as a template. It was found that h-BN mostly nucleated at the junction of SWCNTs  rather tAdditionally, we investigated the effect of growth time on the growth of SWCNT@h-BN. 3 supercells were employed, and BN molecules were subsequently placed on the tube wall with defects, and with a perfect surface (defect − Eperfect, where Edefect is the energy of the model of BN molecules on the defect (perfect is the energy of the model of BN molecules on the perfect surface (To elucidate the defect-induced heteroepitaxial growth mechanism, we performed first-principles calculations to further investigate the role of defects in the nucleation of h-BN. In order to simulate the SWCNT surface, 27 × 27 × 25.37 Å surface . We usede defect a, and Ep surface b. As calSince h-BN nucleates from the defect sites on the wall of SWCNTs, we tried to prepare SWCNT@h-BN film with different microstructures by tuning the defect density. SWCNT films were treated by air plasma for different time of 20 s, 40 s, 60 s, and 100 s with a power of 7 W. Upon prolonged treatment time, more defects were generated on the wall of SWCNTs . Then, CIn summary, SWCNT@h-BN thin films were prepared using SWCNTs with defects as a template. Air plasma treatment was performed to introduce defects that acted as nucleation sites for the CVD growth of h-BN. Combined experiments and first-principles calculations revealed a defect-induced heteroepitaxial growth mechanism of the SWCNT@h-BN. The density of defects on the wall of SWCNTs could be tuned by adjusting the plasma treatment time, and SWCNT@h-BN films with different microstructures were obtained controllably. This work demonstrated that SWCNTs with defects are a promising template for building SWCNT-based derivatives efficiently.


Table 2A.In the published article, there was an error. We stated that in the first six month after vaccination, BCG vaccination in the afternoon offered better protection against SARS-CoV-2 infections than BCG vaccination in the morning. Given the lack of statistical significance in the analysis comparing the BCG-vaccinated individuals to those who received placebo in the morning and afternoon, it is not accurate to use ‘protection’ when describing the significant interaction hazard ratio in A correction has been made to the conclusion of the Abstract. This sentence previously stated:“Vaccination with BCG in the afternoon offered better protection against SARS-CoV-2 infections than BCG vaccination in the morning in the first six months after vaccination.”The corrected sentence appears below:“Although there was a difference in effect between morning and afternoon BCG vaccination, the vaccine did not protect against SARS-COV-2 infections and clinically relevant RTI’s at either timepoint.”A correction has been made to the last three paragraphs of the results. The paragraphs previously stated:Table 2A). The cumulative incidence of SARS-CoV-2 infection in the BCG afternoon group also tended to be lower than in the respective placebo group, although not statistically significant . In the morning, results are in the opposite direction, and the placebo group tended to be better protected against SARS-CoV-2 . In the period from six months after vaccination until 12 months after vaccination cumulative incidences were comparable (Table 2B). The SDHR of SARS-CoV-2 infections was 1.460 (95% CI 0.505-4.223) for the afternoon BCG group and 0.745 (95% CI 0.43-1.600) for the morning BCG group . A better protection of BCG vaccination in the afternoon against SARS-CoV-2 is also reflected in the analysis of the full 12 months follow-up (cumulative incidences 0.035 [95% CI 0.019-0.060] in the afternoon versus 0.067 [95% CI 0.044-0.097] in the morning), but neither statistically significant (Table 2C).“In the first six months after vaccination, the cumulative incidence of SARS-CoV-2 infection was significantly lower in the BCG afternoon group compared to the BCG morning group  (Tables 2A-C).Due to the interventions of the COVID-19 pandemic, such as quarantine, isolation, and social distancing, the number of clinically relevant RTIs was much lower than SARS-CoV-2 infections. The IHR of the morning BCG group was 0.351 (95% CI 0.025-4.978) in the first part of the year, 2.260 (95% CI 0.376-13.571) in the second part of the year and 1.218 (95% CI 0.295-5.037) in the analysis of the full 12 months . In the afternoon results are in the opposite direction, but not statistically significant . When comparing the BCG morning and afternoon group with each other, the interaction hazard ratio [IHR] is 8.966 (95% CI 1.366-58.836), indicating a difference in effect between the two timepoints. In the second part of the year, cumulative incidences were more comparable with SDHRs of 0.745 (95% CI 0.437-1.600) and 1.460 (95% CI 0.505-4.223) for the morning and the afternoon group, respectively (Table 2B). The IHR of the two BCG groups is 0.530 (95% CI 0.149-1.881). The analysis of the full 12 months follow-up is in line with the aforementioned and did not reveal any statistically significant differences in the cumulative incidence of SARS-CoV-2 infection (Table 2C).“In the first six months after vaccination, the cumulative incidence of SARS-CoV-2 infection was 0.014 (95% CI 0.005-0.031) in the placebo morning group and 0.034 (95% CI 0.018-0.056) in the BCG morning group  (Tables 2A–C).Due to the interventions of the COVID-19 pandemic, such as quarantine, isolation, and social distancing, the number of clinically relevant RTIs was much lower than SARS-CoV-2 infections. The SDHR was comparable in all time periods . Possible explanations may be that in the experimental study from de Bree et al. the time period between vaccination and blood collection was just three months, and that the morning group was vaccinated between 8:00 and 9:00 and the afternoon group at 18:00. …”The corrected paragraphs appears below:“The results of the present study show that the time of day of BCG vaccination did not affect the susceptibility to respiratory infections. We observed some differences in the cumulative incidence of SARS-CoV-2 infections, especially in the first six months after vaccination, but the number of events was too low and consequently confidence intervals were too wide to draw any conclusion. Notably, the direction of the effects was even in the opposite direction of our initial hypothesis that BCG vaccination offers better protection in the morning. It is important mentioning that the initial trial was not powered nor designed to analyze the effect of circadian rhythm.The most likely explanation for our findings is that BCG vaccination simply has no effect on the protection against RTIs and SARS-CoV-2 infections in this study. A protective effect has previously been demonstrated in several smaller studies , but pathophysiological differences between SARS-CoV-2 infections and other RTIs (such as influenza) may account for these differential effects of BCG (23). Another explanation why our results contradict those from de Bree et al. may be that in their experiments the time period between vaccination and blood collection was just three months, and that the morning group was vaccinated between 8:00 and 9:00 and the afternoon group at 18:00 (7).”A correction has been made to the third paragraph of the Discussion. The following sentences were removed:“A trend towards lower incidence of SARS-CoV-2 infections in the BCG afternoon group compared to the placebo group in the first six months, may also point to a – if at all – better protection of BCG vaccination in the afternoon. An explanation for the absence of a clear effect may be that BCG vaccination has no effect on the protection against SARS-CoV-2 infections which makes the timing of the vaccination irrelevant.”A correction has been made to the third paragraph of the Discussion. The following sentence was moved further up:“A protective effect has previously been demonstrated in several  studies , but pathophysiological differences between SARS-CoV-2 infections and other RTIs (such as influenza) may account for these differential effects of BCG (23).”A correction has been made to the fourth paragraph of the Discussion, paragraph 4. The following sentence was removed:“Interestingly, we only observed a trend towards protective effects of BCG only in the first six months after vaccination, which could mean that trained immunity effects are relatively short-lasting.”The authors apologize for these errors and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.


There were no correlations between galectins concentrations and psoriasis severity in PASI or disease duration (p > 0.05). Gal-1 and 12 were significantly negatively correlated with GFR  and gal-2 with HDL (p < 0.05). Gal-2 was significantly positively correlated with CRP (p < 0.05) and gal-12 with fasting glucose (p < 0.01). Based on the results and given the reported role of galectins in metabolic disorders we may conclude that gal-1, 2 and 12 could be potentially engaged in metabolic complications in psoriatics, most probably in atherosclerosis. Gal-2 could be perhaps further investigated as a marker of metabolically induced inflammation in psoriasis, gal-1 and gal-12 as predictors of renal impairment in psoriatics due to metabolic disorders. Potentially, gal-12 could be considered in the future as a marker of carbohydrate metabolism disorders in psoriatics.Psoriasis is a complex disease that nowadays is considered not only a dermatosis but a kind of systemic disorder associated with many accompanying diseases. Metabolic complications leading to cardiovascular incidences are the cause of increased mortality in psoriatic patients. Galectins  are beta-galactoside-binding lectins that exert different functions, including engagement in metabolic processes. Our aim was to assess the concentrations of gal-1, 2 and 12 in psoriatics, to establish their potential clinical implications, including in metabolic complications. Plasma galectins were assessed by ELISA in 60 psoriatic patients and 30 controls without dermatoses and a negative family history of psoriasis. Plasma concentrations of all galectins were significantly higher in patients than controls (gal-1 with  Psoriasis is a complex disease that nowadays is considered not only a dermatosis but a kind of systemic disorder associated with many accompanying diseases . To dateGalectins are beta-galactoside-binding lectins and all of them possess at least one carbohydrate recognition domain ,6. GalecLGALS1 gene on chromosome 22q12 [Gal-1 is a prototype galectin , encodedme 22q12 . Gal-1 ime 22q12 . It is pme 22q12 . Gal-1 hme 22q12 . Of noteme 22q12  and inflme 22q12 .LGALS2 gene on chromosome 22 and appears in the form of a dimer [Gal-2, similar to gal-1, is a prototype galectin encoded by the  a dimer ,8,14. ItLGALS12 gene on chromosome 11 [Gal-12 belongs to the tandem repeat type family of galectins  and is eosome 11 . It is eosome 11 . It is fosome 11 . The bioosome 11 . Gal-12 osome 11 ,16. As fThe three galectins we selected are also involved in different metabolic disorders, namely atherosclerosis, carbohydrate metabolism disorders or dyslipidemia ,10,15. T2. Laboratory tests, including C-reactive protein (CRP), complete blood count, plasma fasting glucose, lipid parameters, aminotransferases, creatinine and urea were performed before the study. The study was approved by the Bioethics Committee of the Medical University of Bialystok (number: APK.002.19.2020) and was conducted in accordance with the principles of the Helsinki Declaration [The study enrolled 60 patients (21 women and 39 men) with a flare of plaque psoriasis, with a mean age of 49 ± 2.3 years old. Patients were compared to 30 sex- and age-matched volunteers without dermatoses and a negative family history of psoriasis. All participants signed an informed consent before initiation. There were the following exclusion criteria from the study applied: age under 18 years old, pregnancy, types of psoriasis other than plaque, dietary restrictions, intake of oral medications at least 3 months prior to the study, infectious diseases, autoimmune diseases (other than psoriasis) and malignant neoplasms. The severity of psoriatic skin lesions was assessed by the Psoriasis Area and Severity Index (PASI) and was performed by the same dermatologist in every patient. The group of patients was divided into three subgroups depending on the severity of the disease: PASI I (PASI < 10)—mild psoriasis, PASI II (PASI 10–20)—moderate psoriasis and PASI III (PASI > 20)—severe psoriasis. Patients were also divided into two subgroups according to psoriasis duration: more or less than 15 years. Body mass index (BMI) was calculated as weight/heightlaration .® . Optical density was read at a wavelength of 450 nm. The concentrations were assessed by interpolation from calibration curves prepared with standard samples provided by the manufacturer. All laboratory tests were performed by the same person in standardized laboratory settings.Fasting blood samples were taken using vacuum tubes. They were centrifugated for 10 min at 2000× g. The obtained plasma was stored at −80 °C until further analysis. Laboratory parameters were measured using routine techniques. Gal-1,2 and 12 plasma concentrations were measured with an enzyme-linked immunosorbent assay (ELISA) provided by Cloud Clonep < 0.05.Shapiro–Wilk’s W test of normality was used for data distribution analysis. The normally distributed data were analyzed using Student’s t-test or one-way analysis of variance (ANOVA) and shown as mean ± SD. The non-Gaussian data were presented as median (full range) and analyzed using the non-parametric Mann–Whitney test or Kruskal–Wallis test. The relationships between the examined parameters were assessed with Spearman’s rank test. Statistical analysis was conducted using GraphPad Prism 9.4 software. The differences were deemed statistically significant when Basic characteristics of the patients and the control group are presented in There were no significant differences between patients and controls in terms of gender, age or BMI (NS).p < 0.001)  .After the division of patients according to psoriasis severity expressed by PASI, there were no differences in gal-1 concentration between the subgroups (NS) a.p < 0.001)  b. After < 0.001) c.p = 0.018) and an upward trend towards creatinine concentration (p = 0.08)  .p = 0.028). There were no direct correlations between gal-1 concentrations and PASI, BMI and psoriasis duration (NS).Gal-1 was significantly positively correlated with the age of patients   .After the division of patients according to psoriasis severity expressed by PASI, sex or psoriasis duration, there were no differences in gal-2 concentration between the subgroups (NS) a–c.p = 0.02) and a positive with CRP   .There were no direct correlations between gal-2 concentrations and PASI, BMI, age, gender of patients and psoriasis duration (NS).p < 0.05)  .After the division of patients according to psoriasis severity expressed by PASI, sex or psoriasis duration, there were no differences in gal-12 concentration between the subgroups (NS) a–c.p = 0.009) and a negative with GFR   .p = 0.014). There were no direct correlations between gal-12 concentrations and PASI, BMI and psoriasis duration (NS).Gal-12 was significantly positively correlated with the age of patients . Regarding these galectins that we chose in this experiment, the protein and mRNA of gal-1 were reported by Correa et al. to be increased in patients with AD  and in aAtherosclerosis and psoriasis have much in common. Both depend on chronic inflammation and there are similarities between psoriatic and atherosclerotic plaque with regard to cells engaged in these diseases and the cytokine profile . This isThe first of the galectins that we studied, gal-1, is the best-characterized galectin and has already been studied in psoriasis; however, the aims of the authors were different from ours and analyzed from an immunological point of view . In the In our study, we noticed a significantly higher plasma concentration of gal-1 in psoriatic patients compared to controls without dermatoses indicating its role in this specific group of patients.Gal-1 has been implicated in the pathogenesis of various metabolic disorders. First, it is involved in atherosclerosis. Gal-1 has been found to be upregulated in vascular smooth muscle cells (VSMCs) and monocytes. It has been suggested that gal-1 plays a role in the proliferation, adhesion and migration of these cells . That, oThe second metabolic disorder that gal-1 is associated with, and is the basis of metabolic syndrome at the same time, is obesity. Blood concentrations of gal-1 have been associated with obesity and found to be even positively correlated with BMI and plasma triglyceride concentrations, but negatively with plasma HDL . Gal-1 iAnother component of metabolic syndrome is type 2 DM. Elevated concentrations of gal-1 have been found in individuals suffering from DM and this galectin has even been proposed as a new marker of this disease and its risk factor . Alas, iIn this experiment, we found a negative correlation between plasma gal-1 and GFR, as well as an upward trend toward creatinine concentration. Elevated plasma gal-1 levels have been already reported in a small sample of patients with diabetic nephropathy  and suggAnalyzing different clinical aspects of our patients, we did not observe a direct correlation between gal-1 concentrations, suggesting increased metabolic burden, according to the skin lesions severity or psoriasis duration. In comparison, in the study by ElBably et al., skin gal-1 did not correlate with PASI or BSA either . ImportaAs far as we are concerned, there are no studies on the use of gal-1 inhibitors in psoriasis.BRAP gene is associated with an increased risk of myocardial infarction (MI) [LGALS2 gene has been related to the severity of coronary atherosclerosis, but contrary to BRAP, not with MI [LGALS2 rs7291467, and it was independent of BMI and waist-hip ratio (WHR); this experiment, however, was conducted only in women [Gal-2 is much less studied in general and has never been assessed in psoriasis. As for metabolic disorders, gal-2 expression was found to be correlated with a low arteriogenic response in patients with coronary artery disease . BRCA1-aion (MI) . Moreove with MI ,39. Gal- with MI . Another with MI . In the  with MI . Another with MI . As for in women .In our study, we observed significantly higher gal-2 concentrations in psoriatic patients compared to controls, which could again point to a higher risk of metabolic complications in psoriasis. Gal-2 concentrations were not dependent on any clinical-demographic variables such as sex, age, skin lesions severity or duration and there is no other study in which gal-2 would be assessed with reference to such parameters. Apparently, they do not affect significantly gal-2 plasma concentration. However, we found a negative correlation between gal-2 and HDL concentration which is consistent with its role in the promotion of dyslipidemia, as well as a positive correlation with CRP. It has been documented that gal-2 induces the production of lymphotoxin-a, which has pro-inflammatory properties; therefore, gal-2 also has the ability to indirectly promote tissue inflammation . Gal-2 cHowever, there have been attempts to use gal-2 inhibitors, and to the best of our knowledge, there are no studies on their application in psoriasis.The last galectin assessed in this study was gal-12, which, similar to gal-2, has never been evaluated in psoriatic patients. In this experiment, we found a significantly increased concentration of plasma gal-12 compared to controls. Its main action is the negative regulation of lipolysis . Gal-12 In psoriatic patients, we observed a positive correlation between gal-12 and fasting glucose. Potentially, gal-12 could be considered in the future as a marker of carbohydrate metabolism disorders in psoriatics. Moreover, similar to gal-1, it was negatively correlated with GFR which may implicate its value as a renal function marker in psoriatics with metabolic complications.Andrographis nallamalayana on the imiquimod-induced psoriatic skin in mice, there was an improvement in skin condition [Gal-12 has been suggested as a therapeutic target in insulin resistance, type 2 DM and other metabolic disorders . Hsu et ondition . Howeverondition . Such obGal-1 and gal-12 were positively correlated with the age of patients which emphasizes the greater risk of metabolic complications in older age, including in psoriasis.The main limitation of our study is not enough diverse group of participants with high male predominance, originating from one city and of one ethnicity. In the future, we plan to assess the tissue expression of gal-1, 2 and 12 in psoriatic plaques and non-lesional skin to further explore their role in psoriasis.In this study, we investigated plasma concentrations of gal-1, gal-2 and gal-12 in psoriasis, and we are the first to report on the potential involvement of the latter two in this dermatosis. Our study revealed that the plasma gal-1, 2 and 12 in psoriatics are significantly higher than in patients without dermatoses. Given the involvement of these galectins in metabolic disorders reported in previous studies and that all of the investigated galectins were significantly higher in the plasma of patients compared to controls, this could indicate their potential engagement in metabolic complications in psoriasis. Gal-2 could be perhaps further investigated as a marker of metabolically induced inflammation in psoriasis and gal-1 and gal-12 as predictors of renal impairment in psoriatics due to metabolic disorders. Potentially, gal-12 could be considered in the future as a marker of carbohydrate metabolism disorders in psoriatics.


We propose caution use and supervision when using Molnupiravir, especially when patients are still at risk of spreading virus.N4-hydroxycytidine (NHC), the active compound of the drug Molnupiravir, is incorporated into SARS-CoV-2 RNA, causing false base pairing. The desired result is an “error catastrophe,” but this bears the risk of mutated virus progeny. To address this experimentally, we propagated the initial SARS-CoV-2 strain in the presence of NHC. Deep sequencing revealed numerous NHC-induced mutations and host-cell-adapted virus variants. The presence of the neutralizing nanobody Re5D06 selected for immune escape mutations, in particular p.E484K and p.F490S, which are key mutations of the Beta/Gamma and Omicron-XBB strains, respectively. With NHC treatment, nanobody resistance occurred two passages earlier than without. Thus, within the limitations of this purely   •Molnupiravir might give rise to new SARS-CoV-2 variants when used without precautions•In vitro, the active compound of Molnupiravir gives rise to nanobody-resistant variants•We provide an integrated view on the selection of nanobody-escape mutants Pharmaceutical science; Immunology; Virology; Structural biology Besides an unprecedented vaccination effort, this also raises the urgent need for effective therapies.d-N4-hydroxycytidine (NHC).,,,,,Molnupiravir is orally applicable. Upon resorption and cleavage of an ester bond, the active compound is released, i.e., β-,,,Despite these impressive developments, a drug that causes virus mutations cannot be without concern, and the approval of Molnupiravir for clinical use was heavily debated. One reservation consisted of the possibility that Molnupiravir might also induce mutations in the cellular genome, which would then cause damage to embryos (in case of pregnancy) or perhaps contribute to malignant transformation.,,,,,Another concern, however, was raised more recently: Could Molnupiravir treatment facilitate the occurrence of mutant virus with increased virulencein vitro, recapitulating the treatment of SARS-CoV-2 infection by incubating infected cultured cells with the active compound of Molnupiravir, NHC. The virus population released from such cells indeed contained multiple mutations, as revealed by deep sequencing. Strikingly, two to four passages of this virus pool gave rise to selection of distinct mutants with apparent gains of fitness. The Furin cleavage site within the spike protein was mutated, plausibly contributing to virus replication in Vero cells. Most impressively, however, the rise of immune-escape mutants was recapitulated in this system. When incubating the pool with a potently neutralizing nanobody, resistant virus mutants were rapidly selected that had reproducibly mutated specific residues in the spike protein, most notably p.F490S, p.E484K, or p.G446D. These residues, according to structure analyses and AlphaFold predictions, were major mediators of the spike-nanobody interaction. Hence, at least in an in vitro system, NHC can indeed give rise to virus pools containing readily selectable mutants, with efficient adaptation to the cellular environment and complete resistance to a neutralizing nanobody.In the present study, we tried to model Molnupiravir-induced virus mutagenesis ,Vero E6 cells replicate SARS-CoV-2 with high efficiencyDuring the passage of virus pools in the presence of nanobody, we noticed that the tolerance of the virus toward the nanobody increased. Whereas the initial virus pool was largely neutralized by the nanobody, even 10 nM of the nanobody no longer prevented infection with the virus pool obtained at the end of the selection procedure. We observed this by quantifying both the virus RNA B and the,Before and after the incubation with NHC, we subjected the pool of virus RNA to paired-end RNA sequencing analysis  and S3. To gain further insight into the composition of virus mutations introduced by NHC treatment and/or passaging, we determined stop-gain mutations, i.e., nonsense mutations that prevent the full-length synthesis of the protein corresponding to a particular open reading frame (ORF). Immediately after treatment with NHC, stop-gain mutations were distributed quite evenly across the virus genome. With further passaging, however, the number of such mutations decreased, whereas the few “surviving” stop-gain mutations increased in frequency . When anWe next asked whether different conditions, i.e., presence or absence of a neutralizing nanobody, lead to the accumulation of distinct sets of virus mutants. We compared lower and higher passages in the presence of nanobody with two experiments carried out in parallel for each condition. Comparing the four virus pools that were grown in the presence of the nanobody, we found a high degree of overlap between the identified mutations. On the other hand, when comparing the virus populations obtained with and without nanobody, we found far less overlap of mutations. This was true when restricting our analysis to missense mutations with a high frequency of >20% A–4C but Next, we compared the predominant mutations obtained in the presence or absence of the nanobody against the spike protein. Here, we focused our analysis on mutations that were selected with high frequency under one or several conditions. Mutations were often silent or without obvious functional relevance for the protein, e.g., displaying conserved charge or hydrophobicity . We filt,,Analyzing the accumulated mutations within the spike protein strongly suggested increased virus fitness, in particular concerning its replication despite the presence of an initially neutralizing nanobody. Firstly, however, passaging NHC-treated viruses under any condition led to the accumulation of the mutation p.R682P/Q A. This mMost strikingly, however, selecting the virus pool with the nanobody led to the accumulation of the mutation p.F490S at a frequency of about 80% after prolonged passaging. Of note, p.E484K was also found under such conditions. F490 as well as E484 are both located within the receptor binding domain (RBD) of the spike protein, and they are also at the center of the interaction between the spike and nanobody Re5D06. We thus analyzed the structural consequences of these mutations in more detail, as described in the following.Other accumulating mutations of the spike protein have less obvious explanations B. For inThe mutation p.D198G was also found with increased frequency in the nanobody-selected virus pools. The residue D198 within the N-terminal domain (NTD) of the spike was reported to form a salt bridge contact with residue K462 of the RBD in the “down” conformation.Moreover, growing the virus in the presence of nanobody led to the accumulation of the mutation p.N1187D within the Heptad Region 2 of the S2 domain. The heptad regions are essential for fusion of viral and cellular membranes. HR1 and HR2 undergo dramatic changes in conformation during this process, interacting with the membranes and forming a bundle of six alphahelices.in vitro evolution assays can lead to different solutions to achieve nanobody resistance. When the virus had not been exposed to NHC, selection with nanobody eventually led to resistance, too, albeit at later passages. These resistant virus pools displayed the mutations p.F490S/V and p.E484G, similar to the NHC-treated samples, but they also led to the accumulation of the mutation p.L542R. As outlined in the following, all these mutated residues are located at the interface of the spike protein and the nanobody.Finally, we compared NHC-treated and non-treated virus pools, side by side, as to the selection of nanobody-resistant mutants. The same virus preparation was passaged thrice in parallel, in the presence of NHC or the DMSO control. This was followed by incubations with nanobody Re5D06 A. The viThe indicated mutations were present with high frequencies at passage 7 C. We theIn the presence of nanobody Re5D06, mutations of SARS-CoV-2 were strongly selected that replaced the residues F490, E484, L452, and G446 of the spike protein. Indeed, the selected virus population was almost completely resistant to Re5D06, with no detectable reduction of virus propagation in the presence of this nanobody B and 1C.24. As shown in We have previously reported the structure of the RBD in a complex with nanobody Re5D06The indicated mutations were selected during virus propagation in the presence of nanobody Re5D06. This not only raised the hypothesis that the mutations will disrupt the binding of the nanobody. It also suggested that the mutations should not counteract the binding of the virus to its receptor ACE2 through the spike RBD. The complex of the wild-type RBD with the ACE2 was previously found by cryo-electron microscopy.Next, we sought to interrogate the role of these mutations in the interaction between the RBD and the nanobody in more detail. We used the program AlphaFold, which was established to predict the folding of proteins into tertiary structures with high accuracy.We then subjected the same proteins for modeling, but with the mutations p.G446D, p.L452R, p.E484K, and p.F490S within the RBD. p.G446D did not grossly affect the predicted structure B. HoweveTo further investigate the impact of the most commonly found mutation p.F490S on the interaction between the RBD and ACE2, we modeled the complex of both proteins by AlphaFold, with or without the mutation F490S. The predicted structures of the complex did not grossly deviate from each other regardless of the mutation, and they were in agreement with the experimental structure Figures . AlphaFoThese results should be taken within the limits of a structure prediction algorithm. The use of AlphaFold to model the effects of a single mutation on structures still awaits further evaluation. Limitations when analyzing single proteins in this way were reported,Taken together, direct inspection of the RBD-nanobody structure as well as AlphaFold modeling are each compatible with the following concept: The substitutions p.G446D, p.L452R, p.E484K, and p.F490S allow the RBD to maintain its overall structure, preclude nanobody binding, and preserve ACE2 binding. In agreement, the mutations are also found in naturally occurring variants of SARS-CoV-2, i.e., p.F490S in variant Lambdain vitro system. Even more remarkably, the virus pool obtained in a single flask of NHC-treated cells proved sufficient for the rapid development of cell-adapted and antibody-resistant virus mutants, and NHC considerably accelerated the development of resistance.Our findings not only provide direct evidence that NHC, the active compound of Molnupiravir, is capable of inducing mutations in SARS-CoV-2 in an ,,,Overall, these findings strongly suggest that NHC treatment can give rise to virus mutants with increased fitness. However, our cell culture system is experimental in nature, and it is an open question whether this can be transferred to the situation of a Molnupiravir-treated COVID-19 patient. There can be little doubt that Molnupiravir will also give rise to virus mutants when used in the clinics. Indeed, recent reports provide evidence that such mutations are actually seen in patients.One concern would be that such virus mutants might quickly escape not only the immune response within an individual patient but also the antibody repertoire raised in a vaccinated population. In this context, it seems particularly worrisome that a small NHC-treated culture sample was capable of rapidly evolving resistance to one of the most potently neutralizing antibody structures reported so far, i.e., nanobody Re5D06.Besides immune escape, virus variants may also be selected to accelerate transmission between individuals. This can be achieved by increased stability of the infectious particles, by shifted cell tropism but perhaps most conceivably by enhanced efficiency of the pre-established viral entry mechanism. At least in the experimental system presented here, the most impressive mutagenesis was found within the spike protein. Not only the RBD as such was subject to nanobody-escape mutations. Mutations at the cleavage site of the spike were also found particularly often. Hence, mutations were selected to affect the virus-receptor interactions and the fusion of membranes, and this represents an important opportunity for coronaviruses to vary infectivity and cell tropism. Such mechanisms appear to gain momentum by a drug that mutates the virus genome. Thus, there is a concern that transmissible mutants could occur if Molnupiravir is used inappropriately, especially when treating patients with suboptimal doses, or for insufficient durations, and without isolating them. However, if used with appropriate care, the continued, controlled use of Molnupiravir might still be beneficial.A third concern is that Molnupiravir may promote the resistance of viruses against the drug itself or against additional therapeutics that were applied together with it. Since Molnupiravir does not work through direct inhibition of viral enzymes, Molnupiravir-resistant virus mutants may be unlikely to occur. And indeed, even prolonged NHC treatment did not lead to resistance formation in other coronaviruses.HPRT1-deficient mutants,Beside the mutagenesis in virus genomes, another concern of using Molnupiravir consists in the possible risk of mutagenesis in the DNA of patient cells. Such mutations, if they occur, might give rise to cancer. In pregnant women, they might also cause damage to the embryo. Currently, there is limited data to support such a scenario. Prolonged treatment of cultured cells with NHC for several weeks gave rise to low numbers of Why did we observe the accumulation of different mutations in parallel experiments with seemingly identical conditions? As in the real world, identical conditions can lead to the evolution of different variants of an organism, since stochastic processes play an important role and since several genetic solutions can often be found to meet a phenotypic challenge.,Molnupiravir is not the only drug that develops its antiviral activity through mutations in the virus genome.Further studies are required to assess the occurrence of mutant SARS-CoV-2 in Molnupiravir-treated animals and patients. However, even the available data are raising concerns regarding the use of Molnupiravir outside a controlled clinical setting. We propose that treatment with Molnupiravir should be limited to patients at high risk of developing severe COVID-19. Treatment doses and duration should be chosen to eliminate the virus below detectability. And the patients, while still being infectious, should avoid social contacts to limit the possible spread of virus variants that were formed due to Molnupiravir-induced mutagenesis.in vitro, i.e., in a cell culture system and using a single species of a spike-binding nanobody. This is different from the in vivo situation by several aspects: An infected body replicates the virus with different kinetics and in multiple cell types. These cells are likely to take up Molnupiravir and its metabolites with different efficacies. The immune response comprises a multitude of antibodies, flanked by T cells as well as the natural immune response. Finally, the virus spreads to other individuals through aerosols, not through incubation with tissue culture supernatant. In addition to these considerations, it should also be noted that we deliberately used a suboptimal dose of NHC in our study. Obviously, an NHC dose that eliminates all virus would not give rise to any mutants. Conceivably, keeping drug concentrations well above full efficiency levels would likely reduce the likelihood of raising escape mutants in vivo, too. Thus, the results of our study should be taken as what they are: a strongly simplified model system that reflects only single aspects of the infection of an organism. We show that Molnupiravir has the potential of selecting antibody-resistant virus. It remains to be seen whether this actually happens in a real-world setting, especially when patients are treated with Molnupiravir without fully suppressing virus propagation.The experiments shown here were all performed mdobbel@uni-goettingen.de).Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Matthias Dobbelstein (This study did not generate new unique reagents.2. Vero E6 cells were authenticated in 2021 by means of Cytochrome C Subunit I (COI) DNA Barcoding by the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ). Furthermore, the cells were routinely tested to ensure they were negative for mycoplasma contamination, using the MycoAlert Assay Control Set (Lonza).Vero E6 cells (Vero C1008) were obtained from the German Primate Research Center Göttingen. Cells were maintained in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum , 50 units/mL penicillin, 50 μg/mL streptomycin (Gibco), 2 μg/mL tetracycline (Sigma) and 10 μg/mL ciprofloxacin (Bayer) at 37°C in a humidified atmosphere with 5% CO4-hydroxycytidine  at the concentrations indicated in the legends to 250,000/750,000 cells were seeded into T25/T75 flasks, respectively, using medium containing 2% FBS, and incubated for 8 h at 37°C. Cells were treated with β-D-NFor RNA isolation, 100 μL of the SARS-CoV-2-containing cell culture supernatant was mixed (1:1 ratio) with the Lysis Binding Buffer from the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche) to inactivate the virus. The viral RNA was isolated using Trizol LS, chloroform, and isopropanol. After washing the RNA pellet with ethanol, the isolated RNA was re-suspended in nuclease-free water. Quantitative reverse transcription and polymerase chain reaction (RT-PCR) was performed involving a TaqMan probe,50), the virus-containing supernatant was titrated (endpoint dilution assay). Vero E6 cells were seeded in 96-well plates and incubated with 10-fold dilutions  of virus for 48 h and then fixed with 4% formaldehyde in PBS for 1 h at room temperature. After permeabilization with 0.5% Triton X-100 in PBS for 30 min and blocking in 10% FBS/PBS for 10 min, a primary antibody was used to stain the SARS-CoV-2 Nucleoprotein . The secondary Alexa Fluor 546-coupled donkey anti-rabbit IgG antibody  was added together with 4′,6-diamidino-2-phenylindole (DAPI) for 1.5 h at room temperature. Fluorescence signals were detected by automated microscopy using a Celígo® Imaging cytometer. The titer was determined according to Spearman and Kärber.To determine the Median Tissue Culture Infectious Dose  was used to trim the adapters, followed by removing the contaminating human host reads by kraken2 (version 2.1.2).FastQC (version v0.11.9)https://www.ncbi.nlm.nih.gov/sars-cov-2/. The filtered alteration allele frequency along genome was plotted against the nucleotide positions using R 4.2.2. Mutation lolliplot was made using trackViewer R package.SARS-CoV-2 genome annotation GCF_009858895.2_ASM985889v3_genomic.gff was obtained from Predictions were carried out using AlphaFold-Multimer version 2.1.1 and 2.2.0. The Multiple Sequence Alignments (MSAs) used for the structure inference were built with the standard AlphaFold pipeline. Template modeling was enabled, but only templates that were released before 12–2019 were used. To model the RBD-ACE2 dimer, we also included more recent templates. As AlphaFold only uses single-chain templates and excludes templates with identical sequence, a simple replication of published structures is impossible.Structures were inferred with eight MSA recycling iterations and all five different model parameter sets. After prediction, models were ranked by the pLDDT score for monomers and pTM score for dimers.modelarchive.org) with the accession codes ma-qp22j (RBDwt-Re5D06), ma-0pnx1 (RBD p.G446D-Re5D06), ma-v4odj (RBD p.L452R-Re5D06), ma-jgqs4 (RBD p.E484K-Re5D06), ma-r62mw (RBD p.F490S-Re5D06), ma-lxnac (RBDwt-ACE2), and ma-eh742 (RBD p.F490S-ACE2). Model confidence predictions per residuum (pLDDT-score) are stored in the b-factors column of the .pdb file. PAE-value-plots for each structure are included in the ModelArchive entries.Predictions are available in ModelArchive . The significance of virus load difference between "Ctrl., +Nb" and "+NHC, +Nb" groups within each passage were estimated using Wilcoxon rank-sum test. Statistical details of each experiment can be found in the corresponding figure legend.


The widespread popularity of e-cigarettes is considered an important public health concern. However, only some studies have investigated the prevalence of e-cigarette use in Shanghai, China. Research on the perceived harmfulness of e-cigarettes and public support for e-cigarette regulations in China is limited. This study aimed to estimate e-cigarette awareness, prevalence, and associated factors among adults in Shanghai, China.This study used data from a representative survey conducted in Shanghai, China, in 2019. The survey was conducted at 64 surveillance points in Shanghai, China, using a multistage, stratified, cluster-randomized sampling design, recruiting community-based Chinese adults aged ≥15 years. Based on the principles outlined in the Global Adult Tobacco Survey (GATS) China Project, data were collected by conducting face-to-face interviews in households. Of the 3200 selected households, 3060 people completed the individual survey. The overall response rate was 97.4%.In all, 72.3% of the respondents had heard of e-cigarettes. The respondents who had used e-cigarettes at some point in their life, used them in the last 12 months, and used them currently were 5.8%, 2.6%, and 1.3%, respectively. Among adult residents who had heard of e-cigarettes, 38.2% thought they were less harmful than traditional cigarettes. The respondents who perceived e-cigarettes as more harmful than traditional cigarettes were less likely to have ever used e-cigarettes  and more likely to support incorporating e-cigarettes into the regulation of smoking control .Our findings reveal that the awareness about e-cigarettes was high, and the prevalence of e-cigarette use was similar to the findings from previous studies in China. The harmful perception of e-cigarettes warrants further attention from public health practitioners. E-cigarette sales worldwide are expected to grow to US$28.2 billion by 20273.Electronic cigarettes, also known as ‘e-cigarettes’, are the most common Electronic Nicotine Delivery Systems (ENDS). They are battery-powered devices that deliver smoke-like aerosol (i.e. vapor) that the user inhales6. Chronic exposure to e-cigarette aerosol during pregnancy has potentially harmful developmental effects on offspring8. Growing evidence from clinical and epidemiological studies indicates that e-cigarettes and their aerosol constituents can adversely affect the cardiovascular, brain development, and respiratory systems13.Although the long-term health effects of e-cigarettes remain unclear, sufficient evidence suggests that they are unsafe9.E-cigarettes have become popular as they are perceived to be safer compared to traditional cigarette smoking. This public perception of safety have gained popularity among non-smokers14. Zhao et al.15 identified a significant increase in e-cigarette use among urban populations between 2015 and 2019. Two recent studies used city-level representative data to assess the awareness and use of e-cigarettes among urban residents in China17. However, these studies have yet to report data on e-cigarette use among adults in Shanghai, China. Shanghai is a municipality under the direct administration of the Chinese government. According to the 2020 population census in China, the population of Shanghai was 24.87 million18. The studies conducted in Shanghai may contribute to a better understanding of e-cigarette awareness and use among adult Chinese residents.In China, national survey data showed that e-cigarette use among adults remains low but has increased substantially from 2015 to 20181. If e-cigarettes are not banned, they must be regulated. The Regulation of the Shanghai Municipality on smoking control in public places was officially implemented on 1 March 2010 and was the first domestic tobacco control regulation promulgated by the Provincial People’s Congress. The first amendment to the regulations was implemented on 1 March 2017. Additionally, smoking is prohibited in indoor public places, workplaces, and public transport facilities in Shanghai. On 28 October 2022 inclusion of e-cigarettes within the scope of smoking prohibition in public places in Shanghai was approved and implemented. However, knowledge regarding the characteristics of e-cigarette prevalence and trends in e-cigarette use in Shanghai before e-cigarettes were incorporated into regulations remains limited.Various e-cigarette regulations have been implemented worldwide to reduce the potential risks of e-cigarettes. A total of 32 countries have banned ENDSBased on the knowledge and understanding outlined above, this study aimed to estimate e-cigarette awareness, prevalence, and associated factors among adults in Shanghai. Additionally, we examined the perceived harm and public support for incorporating e-cigarettes into the Regulation of Shanghai Municipality on smoking control to provide important baseline data before the implementing e-cigarette regulation in Shanghai.17. A stratified four-stage cluster sampling design was implemented to produce representative citywide data based on the principles outlined in the GATS Sample Design Manual20.We conducted a cross-sectional household survey in Shanghai, China, from June 2019 to September 2019. Eligible subjects were Chinese residents aged ≥15 years who resided in the selected household in the month prior to the survey date, excluding those collectively living in student dormitories, nursing homes, military camps, prisons, or hospitalsIn the first stage, 64 streets or towns were selected using the probability proportionate to size (PPS) sampling method. The measure of size (MOS) was the number of households. In the second stage, one urban neighborhood community (Ju-Wei-Hui in Mandarin) or rural village was selected from each of the selected streets or towns using the PPS method. The selected secondary sampling unit was partitioned into segments of around 750–1500 households, if the selected unit was of above 1500 households. One segment was randomly selected thereafter. In the third stage, 50 households from each neighborhood community or selected segment, were randomly selected from the household sampling frame created by mapping and listing. The total number of designated households was 3200. In the last stage, one individual was randomly selected from each of the 3200 participating households using simple random sampling.14, with additional questions on the perception of e-cigarette harmfulness and support to incorporate e-cigarettes into the regulation of Shanghai Municipality on smoking control.The questionnaire was based on the Global Adults Tobacco Survey China ProjectSociodemographic characteristics16, annual household income in RMB  and smoking status .Assessed sociodemographic characteristics included: gender, age group , educational level , occupation Measures about e-cigarettes14. Among those who had ever heard of e-cigarettes, the perception of harm around e-cigarettes was measured by the question: ‘Compared with traditional cigarettes, do you think e-cigarettes are less harmful, just as harmful, or more harmful?’. The question: ‘Do you agree with the incorporation of e-cigarettes into the Regulation of Shanghai Municipality on smoking control?’ was asked to learn about the support for e-cigarette regulation in smoke-free venues.Awareness of e-cigarettes was measured by the question: ‘Have you ever heard of e-cigarettes?’. If the answer was ‘yes’, then they were asked ‘Where did you hear about electronic cigarettes?’14.Questions such as ‘Have you ever used an electronic cigarette?’, ‘Have you ever used an electronic cigarette during the past 12 months?’ and ‘Do you use e-cigarettes currently?’ were asked to measure ever use, past 12 months use, and current use of e-cigarettes19. Population data from the Shanghai Municipal Bureau of Statistics were used for the post-stratification calibration adjustment. Descriptive statistics of percentages, including point estimates and 95% confidence intervals (CI), were calculated overall and by sociodemographic characteristics. Rao-Scott chi-squared tests and univariate logistic regression were conducted to compare the outcome variables by sociodemographic characteristics. Multivariable logistic regression was conducted to explore factors associated with awareness and ever use of e-cigarettes. Adjusted odds ratios (AORs) controlled for sociodemographic characteristics, including age, gender, education level, occupation, income, and smoking status, in the analysis of factors associated with awareness of e-cigarettes among all samples. Two models were built to assess factors related to the ever use of e-cigarettes. One model was the same as the AOR analysis of awareness of e-cigarettes. Another model was adjusted for the perceived harmfulness of e-cigarettes compared to traditional cigarettes among respondents who had heard of e-cigarettes, in addition to the factors adjusted in the preceding model. A two-tailed p<0.05 was considered statistically significant.SAS V.9.4 was used for data processing and analysis. Owing to the complex sample design of the survey, each responding unit was assigned a unique survey weight that was used to produce estimates of the population parametersOf the 3200 selected households, 59 empty households were eliminated, 3093 households completed the survey, and 3060 people completed the individual survey. The overall response rate was 97.4%. The weighted percentage estimate of people with senior high school education accounted for 26.5% and those with a college degree or higher were 37.7%. The current smoking prevalence was 19.7% .A total of 72.3% of adults aged ≥15 years had heard of e-cigarettes . AwareneAfter controlling for demographic characteristics and smoking status in Model A, the age groups of 15–24 and 25–44 years were associated with a higher rate of awareness of e-cigarettes. Residents with higher educational level were more likely to be aware of e-cigarette use. Respondents with household incomes ≥50000 had greater odds of being aware of e-cigarettes than those with incomes <30000. Current and former smokers were more likely to be aware of e-cigarettes than non-smokers. There was no association between e-cigarette awareness and gender or occupation .Regarding the information sources about e-cigarettes, the most common source was friends (56.3%), followed by the internet (44.9%) and television (40.7%). The residents who were younger, employed, and had higher level of education were more likely to learn about e-cigarettes on the internet (p<0.0001).Among the adult residents who had heard of e-cigarettes, 38.2% thought that e-cigarettes were less harmful than traditional cigarettes, 24.8% thought they were similarly harmful, and 27.2% were unaware of their harmfulness . More e-Among the respondents who had heard of e-cigarettes, 85.7% supported the incorporation of e-cigarettes into the regulation of the Shanghai Municipality on smoking control . There wAn estimated 1.3% (95% CI 0.6–1.9) of adults aged ≥15 years currently used e-cigarettes, with 2.2% of males and 0.3% of females (p=0.0045). Among the current e-cigarette users, 85.8% used both e-cigarettes and traditional cigarettes. Younger residents were more likely to use e-cigarettes, ranging from 4.1% among people aged 15–24 years to 0.3% among people aged ≥65 years (p=0.0006). Current smokers were more likely to use e-cigarettes than never smokers (p=0.0001) .The prevalence of ever use of e-cigarettes was 5.8% (95% CI: 4.5–7.1), and the prevalence of e-cigarette use in the past 12 months was 2.6% (95% CI: 1.7–3.6), both of which were higher among males than females (p<0.0001). Younger residents were more likely to have used e-cigarettes in the last 12 months and have ever used e-cigarettes . Current and former smokers were more likely to have used e-cigarettes than never smokers (p<0.0001) . After cTo the best of our knowledge, this is the first study to report e-cigarette awareness and prevalence among adults in Shanghai. Perceived harm and public support for incorporating e-cigarettes into the regulation of smoking control in the Shanghai Municipality were also explored. Our study also provides baseline data for evaluating the effect of the amended regulations of Shanghai Municipality on smoking control in public places. The key finding of this study was that those who perceived e-cigarette use as more harmful were less likely to be e-cigarette ever users and more likely to support incorporating e-cigarettes into the regulation of smoking control, compared to individuals who perceived e-cigarette use as less harmful than traditional cigarettes.22. Our study showed a higher level of awareness of e-cigarettes. In the 2018 China Global Adult Tobacco Survey (GATS), the rate of e-cigarette awareness, the prevalence of current e-cigarette use, in the last 12 months, and ever use among urban residents was 56.3%, 1.1%, 2.5%, and 5.3%, respectively14. Results from five representative citywide surveys conducted from 2017 to 2018 revealed that awareness of e-cigarette products was 51.3% among urban adults in five cities, ranging from 45.0% in Chongqing to 58.7% in Xi’an. Furthermore, 4.8% of respondents reported using e-cigarettes, ranging from 3.7% in Wuhan to 6.6% in Xi’an. Approximately 0.9% reported using e-cigarettes in the past 30 days, ranging from 0.6% in Chongqing to 1.7% in Xi’an16. Our study found that among current users of electronic cigarettes, 85.8% were dual users. It was consistent with findings from previous studies in China17, but much higher than that in western populations23. The most likely reason for using e-cigarettes was smoking cessation14.More than 70% of participants in our study were aware of e-cigarette use. The rates of current e-cigarette use, use in the last 12 months, and ever use were 1.3%, 2.6%, and 5.8%, respectively. The prevalence of e-cigarette use in our study was similar to the findings of previous studies in China but lower than the reported prevalence in some developed countries17. These results raise public health concerns among young adults. A report commissioned by Public Health England reveals that 61.9% of youth perceive e-cigarettes as less harmful than cigarettes24. A meta-analysis study showed that, in comparison to non-users, young people who were ever e-cigarette users were two times more likely to perceive e-cigarettes as less harmful than tobacco cigarettes 25. Furthermore, growing research indicates that the use of e-cigarettes may be associated with an increased risk of progression to conventional cigarette smoking in never smokers27. A previous study among university students in Shanghai showed that the most common reason for e-cigarette ever use was ‘less harmful than traditional cigarettes’ (55.0%)28. Our results reveal that the rate of perceiving e-cigarettes as less harmful than traditional cigarettes among young people aged 15–24 years was 55.6%. Moreover, our study found that adult residents who perceived e-cigarettes as more harmful than traditional cigarettes were less likely to have used e-cigarettes than those who perceived e-cigarettes as less harmful. A meta-analysis involving 91051 adolescents and young adults showed that e-cigarette use was associated with a higher risk of smoking intentions27. Another meta-analysis based on 89076 participants showed that the AOR of smoking initiation among e-cigarette users at baseline compared with non-e-cigarette users at baseline was 3.37 (95% CI: 2.68–4.24)29. Previous evidence indicates gateway effects from e-cigarette smoking to conventional cigarette smoking. This suggests that public health practitioners should be concerned about the perception of harm from e-cigarettes to reduce the potential hazards of e-cigarette use.Our study found that the rates of e-cigarette awareness and use were higher among people aged <45 years than among other age groups, similar to the results of previous studies31. Content analysis of the full text of 639 news articles in mainland Chinese newspapers from 2004 to 2019 found that ‘e-cigarettes are less harmful than traditional cigarettes’ is the dominant argument compared with the opposite argument that ‘e-cigarettes are as harmful or more harmful than traditional cigarettes’ within the topics of relative health impact compared to cigarettes31. A previous mixed-methods study on adult e-cigarette users in China found that some participants received e-cigarette related information from news reported by the official media. Some participants saw television or internet news reports stating that e-cigarettes were harmful, thus raising doubts about e-cigarette safety32. Consistent with previous research, our study found that public media, such as the Internet and television, are the main information sources of e-cigarettes in China33. These findings suggest that health educators should provide the public with updated health knowledge about e-cigarettes through popular social media platforms.The media is the most accessible information source for the public, shaping their perceptions of health issues31. Currently, the Shanghai Municipality Regulation on smoking control in public places, bans e-cigarette use in smoke-free venues. Our findings provide strong public support for a ban on e-cigarette use in smoke-free venues in Shanghai. However, more efforts are needed to reinforce the governance of e-cigarette use. Media reports on the harmfulness of e-cigarettes and policies regarding e-cigarettes may underpin the strong support for regulation.Government policies significantly influence Chinese media coverage of relevant issues. The increased reporting of policies unfavorable to e-cigarettes aligns with the growing number of regulations restricting e-cigarettes14. The prevalence of e-cigarette use among people aged 15–24 years, as shown in our study, may have been underestimated. Second, this was a cross-sectional survey, which precludes causal inferences. Third, this study did not assess the factors associated with current e-cigarette use, considering the sample size. Finally, given that the data used in this study were collected in 2019, temporal differences may exist.This study has several limitations. First, our survey was a household survey, excluding those who lived collectively in places such as student dormitories, nursing homes, and military camps; there may be a few college students covered by the surveyOur findings revealed that awareness of e-cigarettes was high, and the prevalence of e-cigarette use in our study was similar to findings from previous studies in China. The harmful perceptions of e-cigarettes warrant further attention from public health practitioners. Our study provides baseline data for the evaluation of the effect of the amended Regulation of the Shanghai Municipality on smoking control in public places. Future studies on e-cigarette use in Shanghai are critical for examining trends in prevalence and the potential effects of regulations.